Desmosomes are the most prominent intercellular junctions in the epidermis, where they serve dual roles in cell-cell adhesion and in providing an attachment site for the intermediate filament (IF) cytoskeleton. The existence of acantholytic autoimmune diseases that target the desmosomal cadherins and inherited diseases that involve perturbations of the desmosomal-IF complex, suggests that desmosomes may be crucial for maintaining epidermal integrity. The most abundant desmosomal components are the desmoplakins (DPs) which are localized at a potentially critical interface between IF and the cytoplasmic domains of the desmosomal cadherins. DPs may therefore be required for integrating the IF cytoskeleton into a supracellular network that joins all cells within the epidermis. In previous work, our laboratory identified and characterized human DP cDNA clones, and subsequently investigated the function of each of the three major DP domains. Transfection studies suggested that the C- terminal globular domain facilitates association with IF networks, the central alpha-helical coiled-coil rod domain promotes assembly of higher order structures resembling the desmosomal plaque, and the N-terminus targets DP to desmosomal plaques. This revised application will focus on the function of these domains and the identity of their binding partners in more detail. Furthermore, the importance of desmosomal integrity during development and epidermal differentiation will be addressed. The specific aims are: 1) to identify DP sequences that are necessary and sufficient for targeting and retention of DP in the desmosomal plaque, 2) to test the hypothesis that the cytoplasmic tails of the desmosomal cadherins (with or without plakoglobin) act as cell surface binding sites for DP, and if these molecules are insufficient, to identify potentially novel binding partners for the DP N-terminus, 3) to define a) the mechanisms by which the DP C-terminus interacts with IF networks and b) how this interaction is modulated by phosphorylation of DP, 4) to determine the importance of desmosomal anchorage of IF by ectopically expressing disrupter constructs that uncouple IF from the plaque in developing Xenopus embryos and in the epidermis of transgenic mice. These experiments will lay the groundwork for understanding how desmosomes and the desmosome-IF attachment is established and how it may be perturbed in acquired and inherited pathologies.